Volume 14, Issue 11, Pages 1207-1214 (November 2007) Inverse In Silico Screening for Identification of Kinase Inhibitor Targets  Stefan Zahler, Simon Tietze, Frank Totzke, Michael Kubbutat, Laurent Meijer, Angelika M. Vollmar, Joannis Apostolakis  Chemistry & Biology  Volume 14, Issue 11, Pages 1207-1214 (November 2007) DOI: 10.1016/j.chembiol.2007.10.010 Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 1 Docking Results and In Vitro Kinase Assay (A) Indirubin 3′-oxime (thick, atom colored sticks) redocked into a CDK5 structure in complex with indirubin 3′-oxime. The resulting score leads to rank 11 for this structure among the kinases and 36 in the complete set of complexes. The model is in perfect agreement with the typical interaction motif of two or three hydrogen bonds (shown as dashed blue lines) to the protein backbone (shown in the background of this picture). The conformation of indirubin 3′-oxime as found in the PDB structure is shown as thin cyan sticks and shows nearly perfect agreement with the model. (B) Indirubin 3′-oxime (thick, atom colored sticks) docked into a CDK2 structure. This complex has rank 1 among the kinases and rank 5 in the complete set. The model is in perfect agreement with the typical interaction motif of two or three hydrogen bonds (shown as dashed blue lines) to the protein backbone (shown in the background of this picture). The ligand originally contained in the X-ray structure is shown as thin cyan sticks for comparison. (C) 6-bromo inidirubin 3′-oxime model into PDK1 with bound inhibitor staurosporine. The figure shows the good agreement with the backbone hydrogen bonding motif. (D) The solvent accessible surface of the binding pocket of PDPK1 together with the VDW-Sphere depiction of 6-bromo inidirubin 3′-oxime indicate that the binding pocket can easily accommodate the halogen. (E) 7-bromo inidirubin 3′-oxime docked into PDK1 with bound inhibitor LY333531. The binding motif is in principle present; however, the geometry of the hydrogen bonds to the backbone is not optimal. (F) Dose-response relationships for the three compounds in the in vitro kinase assay for PDK1. 5BIO and 7BIO show inhibition only at the highest concentration tested (100 μM), whereas 6BIO shows a classical sigmoidal curve with an IC50 of 1.5 μM. (G) 2D structures of the indirubin derivatives. Chemistry & Biology 2007 14, 1207-1214DOI: (10.1016/j.chembiol.2007.10.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 2 Effects of Indirubin Derivatives on Proliferation MCF-7 cells (A–C) and HMEC-1 cells (D–F) were observed after 72 hr growth in the absence or presence of the indicated compounds. Proliferation was measured colorimetrically (OD) by using the crystal violet assay. Error bars denote standard error of the mean taken from three measurements with three replicas each. Chemistry & Biology 2007 14, 1207-1214DOI: (10.1016/j.chembiol.2007.10.010) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 3 Endothelial Cell Migration Is Most Potently Inhibited by 6BIO In the scratch wound, migration assay with endothelial cells complete wound closure is observed in the presence of 10% serum (data not shown). In the absence of serum, the gap remains open (A). 5BIO and 7BIO did only moderately influence migration (B and D), while 6BIO elicited 75% inhibition (C). (E) Quantitative evaluation. Error bars denote standard error of the mean taken from three measurements with three replicas each. (F–H) 6BIO inhibits phosphorylation of PDK1-specific sites in p70S6 kinase (T229) and Akt (T308) in MCF-7 cells. In contrast to 5BIO (F) and 7BIO (H), 6BIO (G) inhibits phosphorylation of PDK1-specific targets (indicated by boxes). An irrelevant phosphorylation site at T389 in p70S6 and overall content of Akt remain unchanged (F–H). Chemistry & Biology 2007 14, 1207-1214DOI: (10.1016/j.chembiol.2007.10.010) Copyright © 2007 Elsevier Ltd Terms and Conditions